{"gene":"LYPD3","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2003,"finding":"C4.4A (LYPD3) was identified as a binding partner for hAG-2 (AGR2) and hAG-3 by yeast two-hybrid cloning, and also binds extracellular alpha-dystroglycan (DAG-1).","method":"Yeast two-hybrid cloning","journal":"British journal of cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single yeast two-hybrid screen, no reciprocal Co-IP validation reported in abstract","pmids":["12592373"],"is_preprint":false},{"year":2004,"finding":"Human C4.4A is a GPI-anchored protein containing two Ly6/uPAR/alpha-neurotoxin modules, extensively modified by 5-6 N-linked and ~15 O-linked carbohydrates; a protease-sensitive region (Tyr200-Arg204) lies between the N- and O-glycosylation clusters. Recombinant soluble C4.4A and GPI-anchored C4.4A from amnion membranes showed no detectable interaction with uPA, distinguishing C4.4A functionally from uPAR.","method":"Recombinant protein expression/purification, mass spectrometry characterization of glycosylation, ELISA-based binding assay, immunohistochemistry","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods (protein purification, glycosylation mapping, binding assay) in single study; negative uPA-binding result rigorously established","pmids":["15012588"],"is_preprint":false},{"year":2004,"finding":"C4.4A does not interact with urokinase-type plasminogen activator (uPA), indicating it lacks functional overlap with its structural homologue uPAR despite shared domain architecture.","method":"ELISA-based binding assay with recombinant soluble C4.4A and MCF7 cells expressing GPI-anchored C4.4A","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with two independent C4.4A preparations (recombinant soluble + natural GPI-anchored); single lab","pmids":["15012588"],"is_preprint":false},{"year":2005,"finding":"C4.4A binds laminin-1 (LN1) and laminin-5 (LN5) as extracellular ligands; C4.4A-expressing tumor cells show increased spreading, lamellipodia formation, and migration on LN5 and LN1. C4.4A also associates with galectin-3, which influences laminin adhesion.","method":"ELISA screening of ECM components with recombinant rat C4.4A, cDNA transfection of BSp73AS cells, cell migration/spreading assays, co-immunoprecipitation for galectin-3 association","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ELISA binding confirmed with transfected cells, functional migration assays; single lab but multiple orthogonal methods","pmids":["15729693"],"is_preprint":false},{"year":2007,"finding":"C4.4A transcription requires C/EBPbeta binding to a TRE/CCAAT composite element (-71 to -88 bp) in the promoter, is enhanced by JunD or c-Jun co-transfection, and the TATA-less GC-rich core promoter alone is insufficient; Sp3 but not Sp1 binding sites are functional.","method":"Reporter construct deletions/point mutations, mobility-shift assays (EMSA), co-transfection studies, chromatin immunoprecipitation (ChIP)","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and EMSA with mutagenesis reporter constructs; single lab, multiple complementary methods","pmids":["17278103"],"is_preprint":false},{"year":2008,"finding":"C4.4A is a substrate for both ADAM10 and ADAM17 metalloproteases, identified by SILAC-based proteomics in MCF7 cells with shRNA knockdown of ADAM10 or ADAM17; cleavage by these proteases likely contributes to tumor invasion.","method":"SILAC proteomics, shRNA knockdown of ADAM10/ADAM17 in MCF7 cells","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics (SILAC) with genetic perturbation; single lab, mechanistic follow-up limited to what is stated in abstract","pmids":["18979631"],"is_preprint":false},{"year":2012,"finding":"C4.4A associates with α6β4 integrin and MT1-MMP (MMP14) as well as TACE in lipid rafts; hypoxia promotes this association and drives a shift from laminin adhesion to cell motility, accompanied by laminin fragmentation. This complex is maintained in exosomes, and shed C4.4A retains laminin-degrading activity.","method":"Co-immunoprecipitation, in vitro wound healing assays, exosome isolation, CoCl2-induced hypoxia model, siRNA/knockdown experiments","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-immunoprecipitation and functional assays; single lab, multiple orthogonal approaches","pmids":["22431918"],"is_preprint":false},{"year":2012,"finding":"C4.4A knockdown in colorectal cancer cells reduces cell invasion (but not proliferation) and correlates with EMT reversal, including increased E-cadherin and decreased vimentin and N-cadherin, placing C4.4A upstream of EMT regulation.","method":"siRNA knockdown, invasion assays, Western blot for EMT markers (E-cadherin, vimentin, N-cadherin) in HCT116 cells","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with defined molecular phenotype readouts; single lab","pmids":["22404718"],"is_preprint":false},{"year":2013,"finding":"C4.4A promotes metastasis by recruiting activated α6β4 integrin into lipid rafts, where C4.4A cooperates with α6β4 and MMP14 (MT1-MMP) to enable focalized matrix degradation; α6β4 also promotes BAD phosphorylation and upregulation of Bcl2/BclXl, mediating drug resistance. C4.4A knockdown in ASML rat pancreatic carcinoma cells strongly reduces metastasis and sensitizes to cisplatin.","method":"Stable shRNA knockdown of C4.4A in ASML cells, intrafootpad metastasis model in rats, co-immunoprecipitation, raft fractionation, PI3K/Akt pathway analysis","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockdown model with defined pathway analysis and multiple biochemical assays; single lab","pmids":["23727360"],"is_preprint":false},{"year":2015,"finding":"Extracellular AGR2 binds C4.4A directly (co-immunoprecipitation from cell lysates and with recombinant proteins); AGR2 signaling through C4.4A requires laminins 1 or 5 and integrin β1 to stimulate PDAC cell proliferation, migration, invasion, and chemoresistance. C4.4A knockdown reduces migration and gemcitabine resistance.","method":"Co-immunoprecipitation from cell lysates, recombinant protein mixing assay, siRNA knockdown, Boyden chamber assays, FACS apoptosis assay, orthotopic xenograft tumor model with blocking monoclonal antibodies","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct protein-protein interaction confirmed by both cell lysate Co-IP and recombinant protein assay, functional siRNA knockdown, in vivo tumor model; multiple orthogonal methods across in vitro and in vivo settings","pmids":["25646014"],"is_preprint":false},{"year":2016,"finding":"C4.4A-deficient mice (gene ablation) develop normally with intact squamous epithelia but show delayed keratinocyte migration in wound closure (incisional skin wound model in male mice) and reduced incidence of invasive lesions in chemically induced bladder carcinomas, establishing C4.4A as a functional promoter of keratinocyte migration and tumor cell invasion in vivo.","method":"Constitutive C4.4A knockout mouse generation, in vivo wound-healing assay, chemical carcinogenesis bladder tumor model","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — definitive loss-of-function genetic model (KO mouse) with specific in vivo phenotypic readouts in two separate assays","pmids":["27169360"],"is_preprint":false},{"year":2017,"finding":"C4.4A knockdown in hepatocellular carcinoma (Huh7 and HepG2) cells reduces migration and invasion but does not affect proliferation or apoptosis, confirming a specific pro-migratory/invasive function of C4.4A.","method":"siRNA knockdown, migration and invasion assays, proliferation and apoptosis assays","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with defined functional readouts in two cell lines; single lab","pmids":["29048672"],"is_preprint":false},{"year":2017,"finding":"C4.4A (LYPD3) is an internalizing cell surface protein; an ADC targeting C4.4A (BAY 1129980) demonstrates selective antiproliferative activity in C4.4A-expressing cells and requires C4.4A surface expression for efficacy, confirming receptor-mediated internalization.","method":"ADC efficacy assay in C4.4A-transfected vs. non-transfected cells, xenograft and PDX tumor models","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ADC selectivity assay with transfected control cells establishes internalization function; in vivo xenograft data; single lab","pmids":["28292941"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of the two LU domains of human C4.4A were solved (alone and in complex with a Fab fragment of a monoclonal anti-C4.4A antibody). The structure reveals that C4.4A forms a compact globule with both LU domains packed face-to-face, contrasting with the flexible arrangement typical of most LU-domain proteins. The Fab combining site spans both LU domains and overlaps with the AGR2-binding site.","method":"X-ray crystallography (crystal structures of C4.4A D1D2 domains and C4.4A–Fab complex)","journal":"International journal of biological sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination with functional epitope mapping; multiple structural observations confirmed by independent Fab complex structure","pmids":["32140067"],"is_preprint":false},{"year":2020,"finding":"β2-adrenoceptor (ADRβ2) signalling upregulates LYPD3 protein expression in breast cancer cells; LYPD3 knockdown significantly reduces both basal and norepinephrine-induced migration activity of MCF-7 cells, placing LYPD3 downstream of β2-adrenoceptor signalling as a pro-migratory effector.","method":"Protein profiling (mass spectrometry), siRNA knockdown, migration assays, adrenoceptor agonist/antagonist treatment","journal":"Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein-level upregulation confirmed by profiling, functional siRNA knockdown with migration readout; single lab","pmids":["32098331"],"is_preprint":false},{"year":2024,"finding":"Glycosylation of LYPD3 modulates its subcellular localization and reinforces its role in suppressing HNSCC metastasis; miR-151-5p directly targets the 3'-UTR of LYPD3 mRNA to reduce LYPD3 expression, and this targeting is facilitated by METTL3-mediated m6A modification and hnRNP U-mediated miR-151-5p maturation.","method":"3'-UTR luciferase reporter assays, m6A modification analysis, glycosylation experiments with subcellular fractionation/localization, miR-151-5p overexpression and LYPD3 knockdown functional assays","journal":"Molecular biomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct 3'-UTR targeting confirmed, glycosylation-localization link established experimentally; single lab, multiple methods","pmids":["39009906"],"is_preprint":false}],"current_model":"LYPD3/C4.4A is a GPI-anchored, two-LU-domain cell surface protein (crystal structure resolved) that adopts a compact face-to-face LU-domain arrangement, is heavily N- and O-glycosylated (glycosylation regulates its subcellular localization), binds laminin-1 and laminin-5 as extracellular ligands, and directly interacts with AGR2 (at a site spanning both LU domains) to signal in an autocrine loop requiring laminins and integrin β1; it is cleaved by ADAM10 and ADAM17, recruits activated α6β4 integrin and MT1-MMP into lipid rafts to drive focalized matrix degradation and drug resistance, promotes EMT and cell migration downstream of β2-adrenoceptor signalling, and its transcription requires C/EBPbeta binding to a TRE/CCAAT element cooperating with JunD/c-Jun—collectively establishing LYPD3 as a pro-invasive, pro-metastatic scaffold/adaptor whose loss in knockout mice selectively impairs keratinocyte wound-healing migration and reduces invasive tumor formation."},"narrative":{"mechanistic_narrative":"LYPD3 (C4.4A) is a GPI-anchored, two-domain Ly6/uPAR (LU)-module cell surface protein that functions as a pro-invasive, pro-metastatic scaffold coordinating laminin engagement, integrin signalling, and focalized matrix degradation [PMID:15729693, PMID:23727360]. Crystallography shows its two LU domains pack face-to-face into a compact globule, atypical for the flexible LU-domain family, with the AGR2-binding site spanning both domains [PMID:32140067]. It is heavily N- and O-glycosylated, and glycosylation governs its subcellular localization [PMID:15012588, PMID:39009906]. Functionally, C4.4A binds the extracellular ligands laminin-1 and laminin-5 and drives spreading, lamellipodia formation, and migration on these substrates [PMID:15729693]; despite sharing domain architecture with uPAR, it does not bind uPA, marking a distinct functional identity [PMID:15012588]. At the cell surface it recruits activated alpha6beta4 integrin together with MT1-MMP (MMP14) into lipid rafts to enable localized laminin/matrix degradation and, via alpha6beta4-dependent BAD phosphorylation and Bcl2/BclXl upregulation, drug resistance; this complex persists in exosomes and shed C4.4A retains laminin-degrading activity [PMID:22431918, PMID:23727360]. Extracellular AGR2 binds C4.4A directly to drive an autocrine signalling loop requiring laminin-1/5 and integrin beta1 that promotes proliferation, migration, invasion, and chemoresistance [PMID:25646014]. C4.4A acts upstream of EMT, with knockdown reversing mesenchymal marker expression and reducing invasion across multiple carcinoma types [PMID:22404718, PMID:29048672]. Genetic ablation in mice confirms a physiological role: knockout animals develop normally but show delayed keratinocyte wound-closure migration and reduced invasive bladder carcinoma [PMID:27169360]. Its expression is controlled transcriptionally by C/EBPbeta cooperating with JunD/c-Jun [PMID:17278103] and post-transcriptionally by miR-151-5p targeting [PMID:39009906], and it is cleaved by ADAM10 and ADAM17 [PMID:18979631].","teleology":[{"year":2003,"claim":"Established the first candidate binding partners for C4.4A, nominating AGR2/AG-3 and alpha-dystroglycan as interactors and framing it as a partner-engaging surface protein.","evidence":"Yeast two-hybrid cloning","pmids":["12592373"],"confidence":"Low","gaps":["Single yeast two-hybrid screen without reciprocal Co-IP validation","Functional consequence of binding not addressed","alpha-dystroglycan interaction not pursued in later timeline entries"]},{"year":2004,"claim":"Defined C4.4A as a GPI-anchored, heavily glycosylated two-LU-module protein and showed it does NOT bind uPA, distinguishing it functionally from its structural homologue uPAR.","evidence":"Recombinant protein purification, glycosylation mass spectrometry, and ELISA binding with soluble and GPI-anchored C4.4A","pmids":["15012588"],"confidence":"Medium","gaps":["Did not identify the functional ligand of C4.4A","Role of the protease-sensitive Tyr200-Arg204 region not tested functionally"]},{"year":2005,"claim":"Identified laminin-1 and laminin-5 as bona fide extracellular ligands and linked C4.4A to cell spreading and migration on laminin, giving the protein a concrete adhesion/motility function.","evidence":"ELISA ECM screening, cDNA transfection, migration/spreading assays, and galectin-3 Co-IP","pmids":["15729693"],"confidence":"Medium","gaps":["Mechanism by which laminin binding drives migration not resolved","Role of galectin-3 association not mechanistically detailed"]},{"year":2007,"claim":"Resolved how C4.4A transcription is controlled, showing C/EBPbeta binding to a TRE/CCAAT element cooperating with JunD/c-Jun drives expression.","evidence":"Reporter deletion/mutation constructs, EMSA, co-transfection, and ChIP","pmids":["17278103"],"confidence":"Medium","gaps":["Upstream signals controlling C/EBPbeta engagement not defined","Single-lab promoter analysis"]},{"year":2008,"claim":"Identified ADAM10 and ADAM17 as the proteases that cleave C4.4A, providing a mechanism for its shedding during invasion.","evidence":"SILAC proteomics with shRNA knockdown of ADAM10/ADAM17 in MCF7 cells","pmids":["18979631"],"confidence":"Medium","gaps":["Cleavage site and fate of shed fragment not mapped","Functional consequence of shedding inferred rather than directly tested"]},{"year":2012,"claim":"Placed C4.4A in a lipid-raft complex with alpha6beta4 integrin, MT1-MMP, and TACE that switches cells from laminin adhesion to motility under hypoxia and degrades laminin, including from exosomes.","evidence":"Reciprocal Co-IP, wound-healing assays, exosome isolation, and CoCl2 hypoxia model with knockdown","pmids":["22431918"],"confidence":"Medium","gaps":["Stoichiometry and assembly order of the raft complex unknown","Single-lab evidence"]},{"year":2012,"claim":"Positioned C4.4A upstream of EMT, showing knockdown reverses mesenchymal marker expression and reduces invasion without affecting proliferation.","evidence":"siRNA knockdown with EMT marker Western blots and invasion assays in HCT116 cells","pmids":["22404718"],"confidence":"Medium","gaps":["Signalling link between C4.4A and EMT transcriptional program not defined","Single cell line"]},{"year":2013,"claim":"Demonstrated in vivo that C4.4A recruits activated alpha6beta4 integrin into rafts to drive focalized matrix degradation and metastasis, while alpha6beta4-BAD-Bcl2 signalling confers drug resistance.","evidence":"Stable shRNA knockdown in ASML rat carcinoma cells, intrafootpad metastasis model, raft fractionation, and PI3K/Akt pathway analysis","pmids":["23727360"],"confidence":"Medium","gaps":["Direct vs. indirect nature of C4.4A-integrin coupling not fully resolved","Single in vivo model system"]},{"year":2015,"claim":"Confirmed a direct AGR2-C4.4A interaction and defined an autocrine signalling loop requiring laminins and integrin beta1 that drives PDAC proliferation, invasion, and chemoresistance.","evidence":"Co-IP from lysates and recombinant protein mixing, siRNA knockdown, Boyden chamber and apoptosis assays, and orthotopic xenografts with blocking antibodies","pmids":["25646014"],"confidence":"High","gaps":["Downstream effectors of AGR2-C4.4A signalling not fully mapped","How the loop integrates with the alpha6beta4/MMP14 raft complex unclear"]},{"year":2016,"claim":"Provided definitive genetic loss-of-function evidence that C4.4A promotes keratinocyte migration and tumor invasion in vivo.","evidence":"Constitutive C4.4A knockout mice in incisional wound-healing and chemical bladder carcinogenesis models","pmids":["27169360"],"confidence":"High","gaps":["Molecular basis of the wound-healing migration defect not dissected in vivo","Normal development indicates functional redundancy not explored"]},{"year":2017,"claim":"Generalized the pro-migratory/invasive function to hepatocellular carcinoma and validated C4.4A as an internalizing target amenable to antibody-drug conjugate therapy.","evidence":"siRNA knockdown with migration/invasion/proliferation assays in Huh7/HepG2, and ADC (BAY 1129980) efficacy in transfected cells and xenograft/PDX models","pmids":["29048672","28292941"],"confidence":"Medium","gaps":["Internalization route and recycling not characterized","Single-lab functional studies"]},{"year":2020,"claim":"Solved the structure of the two LU domains, revealing an atypical compact face-to-face arrangement and mapping the AGR2-binding/antibody epitope spanning both domains.","evidence":"X-ray crystallography of C4.4A D1D2 alone and in complex with an anti-C4.4A Fab","pmids":["32140067"],"confidence":"High","gaps":["Structure of full-length glycosylated C4.4A and its complexes with laminin/integrin not solved","Conformational dynamics in membrane context unknown"]},{"year":2020,"claim":"Placed LYPD3 downstream of beta2-adrenoceptor signalling as a pro-migratory effector in breast cancer.","evidence":"Mass spectrometry protein profiling, siRNA knockdown, and migration assays with adrenoceptor agonist/antagonist treatment in MCF-7 cells","pmids":["32098331"],"confidence":"Medium","gaps":["Signalling intermediates linking ADRbeta2 to LYPD3 induction not defined","Single cell line"]},{"year":2024,"claim":"Established post-transcriptional control of LYPD3 by miR-151-5p (facilitated by METTL3 m6A and hnRNP U) and linked glycosylation to its subcellular localization and a metastasis-suppressive role in HNSCC.","evidence":"3'-UTR luciferase reporters, m6A analysis, glycosylation subcellular fractionation, and miR/knockdown functional assays","pmids":["39009906"],"confidence":"Medium","gaps":["Apparent context-dependent metastasis suppression contrasts with pro-invasive role in other tissues and is unresolved","Glycoform-specific localization mechanism not detailed"]},{"year":null,"claim":"How the AGR2 autocrine loop, the alpha6beta4/MMP14 raft degradation complex, and the context-dependent pro- versus anti-metastatic roles of LYPD3 are mechanistically integrated remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking signalling input, complex assembly, and tissue-specific outcome","Structure of full-length protein with physiological ligands absent","Direct catalytic activity of LYPD3 itself not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6,8,9]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,6,12]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[6,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,14]}],"complexes":["C4.4A-alpha6beta4 integrin-MT1-MMP/TACE lipid raft complex"],"partners":["AGR2","ITGB4","ITGA6","MMP14","ADAM10","ADAM17","LGALS3","ITGB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95274","full_name":"Ly6/PLAUR domain-containing protein 3","aliases":["GPI-anchored metastasis-associated protein C4.4A homolog","Matrigel-induced gene C4 protein","MIG-C4"],"length_aa":346,"mass_kda":36.0,"function":"Supports cell migration. May be involved in urothelial cell-matrix interactions. May be involved in tumor progression","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O95274/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LYPD3","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LYPD3","total_profiled":1310},"omim":[{"mim_id":"609484","title":"LY6/PLAUR DOMAIN-CONTAINING PROTEIN 3; LYPD3","url":"https://www.omim.org/entry/609484"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"cervix","ntpm":347.9},{"tissue":"esophagus","ntpm":1070.5},{"tissue":"skin 1","ntpm":661.0},{"tissue":"vagina","ntpm":483.6}],"url":"https://www.proteinatlas.org/search/LYPD3"},"hgnc":{"alias_symbol":["C4.4A"],"prev_symbol":[]},"alphafold":{"accession":"O95274","domains":[{"cath_id":"2.10.60.10","chopping":"31-216","consensus_level":"medium","plddt":93.1902,"start":31,"end":216}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95274","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95274-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95274-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LYPD3","jax_strain_url":"https://www.jax.org/strain/search?query=LYPD3"},"sequence":{"accession":"O95274","fasta_url":"https://rest.uniprot.org/uniprotkb/O95274.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95274/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95274"}},"corpus_meta":[{"pmid":"12592373","id":"PMC_12592373","title":"hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan.","date":"2003","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/12592373","citation_count":158,"is_preprint":false},{"pmid":"25646014","id":"PMC_25646014","title":"New Blocking Antibodies against Novel AGR2-C4.4A Pathway Reduce Growth and Metastasis of Pancreatic Tumors and Increase Survival in Mice.","date":"2015","source":"Molecular cancer therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/25646014","citation_count":59,"is_preprint":false},{"pmid":"15012588","id":"PMC_15012588","title":"Structural analysis and tissue localization of human C4.4A: a protein homologue of the urokinase receptor.","date":"2004","source":"The Biochemical 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LYPD3.","date":"2020","source":"Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32098331","citation_count":31,"is_preprint":false},{"pmid":"18979631","id":"PMC_18979631","title":"Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17.","date":"2008","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18979631","citation_count":31,"is_preprint":false},{"pmid":"11180013","id":"PMC_11180013","title":"Upregulation of C4.4A expression during progression of melanoma.","date":"2001","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/11180013","citation_count":29,"is_preprint":false},{"pmid":"22404718","id":"PMC_22404718","title":"C4.4A is associated with tumor budding and epithelial-mesenchymal transition of colorectal cancer.","date":"2012","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/22404718","citation_count":25,"is_preprint":false},{"pmid":"21339181","id":"PMC_21339181","title":"Expression of C4.4A, a structural uPAR homolog, reflects squamous epithelial differentiation in the adult mouse and during embryogenesis.","date":"2011","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/21339181","citation_count":18,"is_preprint":false},{"pmid":"26885441","id":"PMC_26885441","title":"C4.4A as a biomarker of head and neck squamous cell carcinoma and correlated with epithelial mesenchymal transition.","date":"2015","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/26885441","citation_count":17,"is_preprint":false},{"pmid":"21792890","id":"PMC_21792890","title":"Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma.","date":"2011","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/21792890","citation_count":17,"is_preprint":false},{"pmid":"39009906","id":"PMC_39009906","title":"m6A-dependent mature miR-151-5p accelerates the malignant process of HNSCC by targeting LYPD3.","date":"2024","source":"Molecular biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/39009906","citation_count":12,"is_preprint":false},{"pmid":"23727360","id":"PMC_23727360","title":"The metastasis-associated molecule C4.4A promotes tissue invasion and anchorage independence by associating with the alpha6beta4 integrin.","date":"2013","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/23727360","citation_count":12,"is_preprint":false},{"pmid":"17278103","id":"PMC_17278103","title":"CEBPbeta, JunD and c-Jun contribute to the transcriptional activation of the metastasis-associated C4.4A gene.","date":"2007","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/17278103","citation_count":12,"is_preprint":false},{"pmid":"35360840","id":"PMC_35360840","title":"LYPD3, a New Biomarker and Therapeutic Target for Acute Myelogenous Leukemia.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35360840","citation_count":11,"is_preprint":false},{"pmid":"27169360","id":"PMC_27169360","title":"C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27169360","citation_count":11,"is_preprint":false},{"pmid":"25414274","id":"PMC_25414274","title":"Expression of the Ly6/uPAR-domain proteins C4.4A and Haldisin in non-invasive and invasive skin lesions.","date":"2014","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/25414274","citation_count":10,"is_preprint":false},{"pmid":"37924160","id":"PMC_37924160","title":"LY6/PLAUR domain containing 3 (LYPD3) maintains melanoma cell stemness and mediates an immunosuppressive microenvironment.","date":"2023","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/37924160","citation_count":9,"is_preprint":false},{"pmid":"25302166","id":"PMC_25302166","title":"C4.4A as a biomarker in pulmonary adenocarcinoma and squamous cell carcinoma.","date":"2014","source":"World journal of clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/25302166","citation_count":7,"is_preprint":false},{"pmid":"32140067","id":"PMC_32140067","title":"Crystal Structures of Human C4.4A Reveal the Unique Association of Ly6/uPAR/α-neurotoxin Domain.","date":"2020","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32140067","citation_count":6,"is_preprint":false},{"pmid":"23175173","id":"PMC_23175173","title":"Distinct expression of C4.4A in colorectal cancer detected by different antibodies.","date":"2012","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/23175173","citation_count":5,"is_preprint":false},{"pmid":"16776998","id":"PMC_16776998","title":"[Expression and diagnostic application of C4.4A protein in squamous cell carcinoma and adenocarcinoma].","date":"2006","source":"Zhonghua bing li xue za zhi = Chinese journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/16776998","citation_count":4,"is_preprint":false},{"pmid":"29048672","id":"PMC_29048672","title":"De novo synthesis of C4.4A in hepatocellular carcinoma promotes migration and invasion of tumor cells.","date":"2017","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/29048672","citation_count":3,"is_preprint":false},{"pmid":"29075641","id":"PMC_29075641","title":"Expression of C4.4A in an In Vitro Human Tissue-Engineered Skin Model.","date":"2017","source":"BioMed research international","url":"https://pubmed.ncbi.nlm.nih.gov/29075641","citation_count":2,"is_preprint":false},{"pmid":"28777093","id":"PMC_28777093","title":"Expression and crystallographic studies of the D1D2 domains of C4.4A, a homologous protein to the urokinase receptor.","date":"2017","source":"Acta crystallographica. Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/28777093","citation_count":1,"is_preprint":false},{"pmid":"41553571","id":"PMC_41553571","title":"Exploring the molecular function of LYPD3 from pan-cancer to lung cancer: based on bioinformatics and cellular experiments.","date":"2026","source":"Mammalian genome : official journal of the International Mammalian Genome Society","url":"https://pubmed.ncbi.nlm.nih.gov/41553571","citation_count":0,"is_preprint":false},{"pmid":"27634111","id":"PMC_27634111","title":"[Corrigendum] Distinct expression of C4.4A in colorectal cancer detected by different antibodies.","date":"2016","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/27634111","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19356,"output_tokens":4088,"usd":0.059694,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11884,"output_tokens":4511,"usd":0.086097,"stage2_stop_reason":"end_turn"},"total_usd":0.145791,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"C4.4A (LYPD3) was identified as a binding partner for hAG-2 (AGR2) and hAG-3 by yeast two-hybrid cloning, and also binds extracellular alpha-dystroglycan (DAG-1).\",\n      \"method\": \"Yeast two-hybrid cloning\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single yeast two-hybrid screen, no reciprocal Co-IP validation reported in abstract\",\n      \"pmids\": [\"12592373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human C4.4A is a GPI-anchored protein containing two Ly6/uPAR/alpha-neurotoxin modules, extensively modified by 5-6 N-linked and ~15 O-linked carbohydrates; a protease-sensitive region (Tyr200-Arg204) lies between the N- and O-glycosylation clusters. Recombinant soluble C4.4A and GPI-anchored C4.4A from amnion membranes showed no detectable interaction with uPA, distinguishing C4.4A functionally from uPAR.\",\n      \"method\": \"Recombinant protein expression/purification, mass spectrometry characterization of glycosylation, ELISA-based binding assay, immunohistochemistry\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods (protein purification, glycosylation mapping, binding assay) in single study; negative uPA-binding result rigorously established\",\n      \"pmids\": [\"15012588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"C4.4A does not interact with urokinase-type plasminogen activator (uPA), indicating it lacks functional overlap with its structural homologue uPAR despite shared domain architecture.\",\n      \"method\": \"ELISA-based binding assay with recombinant soluble C4.4A and MCF7 cells expressing GPI-anchored C4.4A\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with two independent C4.4A preparations (recombinant soluble + natural GPI-anchored); single lab\",\n      \"pmids\": [\"15012588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"C4.4A binds laminin-1 (LN1) and laminin-5 (LN5) as extracellular ligands; C4.4A-expressing tumor cells show increased spreading, lamellipodia formation, and migration on LN5 and LN1. C4.4A also associates with galectin-3, which influences laminin adhesion.\",\n      \"method\": \"ELISA screening of ECM components with recombinant rat C4.4A, cDNA transfection of BSp73AS cells, cell migration/spreading assays, co-immunoprecipitation for galectin-3 association\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ELISA binding confirmed with transfected cells, functional migration assays; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15729693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"C4.4A transcription requires C/EBPbeta binding to a TRE/CCAAT composite element (-71 to -88 bp) in the promoter, is enhanced by JunD or c-Jun co-transfection, and the TATA-less GC-rich core promoter alone is insufficient; Sp3 but not Sp1 binding sites are functional.\",\n      \"method\": \"Reporter construct deletions/point mutations, mobility-shift assays (EMSA), co-transfection studies, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and EMSA with mutagenesis reporter constructs; single lab, multiple complementary methods\",\n      \"pmids\": [\"17278103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"C4.4A is a substrate for both ADAM10 and ADAM17 metalloproteases, identified by SILAC-based proteomics in MCF7 cells with shRNA knockdown of ADAM10 or ADAM17; cleavage by these proteases likely contributes to tumor invasion.\",\n      \"method\": \"SILAC proteomics, shRNA knockdown of ADAM10/ADAM17 in MCF7 cells\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics (SILAC) with genetic perturbation; single lab, mechanistic follow-up limited to what is stated in abstract\",\n      \"pmids\": [\"18979631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"C4.4A associates with α6β4 integrin and MT1-MMP (MMP14) as well as TACE in lipid rafts; hypoxia promotes this association and drives a shift from laminin adhesion to cell motility, accompanied by laminin fragmentation. This complex is maintained in exosomes, and shed C4.4A retains laminin-degrading activity.\",\n      \"method\": \"Co-immunoprecipitation, in vitro wound healing assays, exosome isolation, CoCl2-induced hypoxia model, siRNA/knockdown experiments\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-immunoprecipitation and functional assays; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"22431918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"C4.4A knockdown in colorectal cancer cells reduces cell invasion (but not proliferation) and correlates with EMT reversal, including increased E-cadherin and decreased vimentin and N-cadherin, placing C4.4A upstream of EMT regulation.\",\n      \"method\": \"siRNA knockdown, invasion assays, Western blot for EMT markers (E-cadherin, vimentin, N-cadherin) in HCT116 cells\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with defined molecular phenotype readouts; single lab\",\n      \"pmids\": [\"22404718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"C4.4A promotes metastasis by recruiting activated α6β4 integrin into lipid rafts, where C4.4A cooperates with α6β4 and MMP14 (MT1-MMP) to enable focalized matrix degradation; α6β4 also promotes BAD phosphorylation and upregulation of Bcl2/BclXl, mediating drug resistance. C4.4A knockdown in ASML rat pancreatic carcinoma cells strongly reduces metastasis and sensitizes to cisplatin.\",\n      \"method\": \"Stable shRNA knockdown of C4.4A in ASML cells, intrafootpad metastasis model in rats, co-immunoprecipitation, raft fractionation, PI3K/Akt pathway analysis\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockdown model with defined pathway analysis and multiple biochemical assays; single lab\",\n      \"pmids\": [\"23727360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Extracellular AGR2 binds C4.4A directly (co-immunoprecipitation from cell lysates and with recombinant proteins); AGR2 signaling through C4.4A requires laminins 1 or 5 and integrin β1 to stimulate PDAC cell proliferation, migration, invasion, and chemoresistance. C4.4A knockdown reduces migration and gemcitabine resistance.\",\n      \"method\": \"Co-immunoprecipitation from cell lysates, recombinant protein mixing assay, siRNA knockdown, Boyden chamber assays, FACS apoptosis assay, orthotopic xenograft tumor model with blocking monoclonal antibodies\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct protein-protein interaction confirmed by both cell lysate Co-IP and recombinant protein assay, functional siRNA knockdown, in vivo tumor model; multiple orthogonal methods across in vitro and in vivo settings\",\n      \"pmids\": [\"25646014\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"C4.4A-deficient mice (gene ablation) develop normally with intact squamous epithelia but show delayed keratinocyte migration in wound closure (incisional skin wound model in male mice) and reduced incidence of invasive lesions in chemically induced bladder carcinomas, establishing C4.4A as a functional promoter of keratinocyte migration and tumor cell invasion in vivo.\",\n      \"method\": \"Constitutive C4.4A knockout mouse generation, in vivo wound-healing assay, chemical carcinogenesis bladder tumor model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — definitive loss-of-function genetic model (KO mouse) with specific in vivo phenotypic readouts in two separate assays\",\n      \"pmids\": [\"27169360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"C4.4A knockdown in hepatocellular carcinoma (Huh7 and HepG2) cells reduces migration and invasion but does not affect proliferation or apoptosis, confirming a specific pro-migratory/invasive function of C4.4A.\",\n      \"method\": \"siRNA knockdown, migration and invasion assays, proliferation and apoptosis assays\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with defined functional readouts in two cell lines; single lab\",\n      \"pmids\": [\"29048672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"C4.4A (LYPD3) is an internalizing cell surface protein; an ADC targeting C4.4A (BAY 1129980) demonstrates selective antiproliferative activity in C4.4A-expressing cells and requires C4.4A surface expression for efficacy, confirming receptor-mediated internalization.\",\n      \"method\": \"ADC efficacy assay in C4.4A-transfected vs. non-transfected cells, xenograft and PDX tumor models\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ADC selectivity assay with transfected control cells establishes internalization function; in vivo xenograft data; single lab\",\n      \"pmids\": [\"28292941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of the two LU domains of human C4.4A were solved (alone and in complex with a Fab fragment of a monoclonal anti-C4.4A antibody). The structure reveals that C4.4A forms a compact globule with both LU domains packed face-to-face, contrasting with the flexible arrangement typical of most LU-domain proteins. The Fab combining site spans both LU domains and overlaps with the AGR2-binding site.\",\n      \"method\": \"X-ray crystallography (crystal structures of C4.4A D1D2 domains and C4.4A–Fab complex)\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determination with functional epitope mapping; multiple structural observations confirmed by independent Fab complex structure\",\n      \"pmids\": [\"32140067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"β2-adrenoceptor (ADRβ2) signalling upregulates LYPD3 protein expression in breast cancer cells; LYPD3 knockdown significantly reduces both basal and norepinephrine-induced migration activity of MCF-7 cells, placing LYPD3 downstream of β2-adrenoceptor signalling as a pro-migratory effector.\",\n      \"method\": \"Protein profiling (mass spectrometry), siRNA knockdown, migration assays, adrenoceptor agonist/antagonist treatment\",\n      \"journal\": \"Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein-level upregulation confirmed by profiling, functional siRNA knockdown with migration readout; single lab\",\n      \"pmids\": [\"32098331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Glycosylation of LYPD3 modulates its subcellular localization and reinforces its role in suppressing HNSCC metastasis; miR-151-5p directly targets the 3'-UTR of LYPD3 mRNA to reduce LYPD3 expression, and this targeting is facilitated by METTL3-mediated m6A modification and hnRNP U-mediated miR-151-5p maturation.\",\n      \"method\": \"3'-UTR luciferase reporter assays, m6A modification analysis, glycosylation experiments with subcellular fractionation/localization, miR-151-5p overexpression and LYPD3 knockdown functional assays\",\n      \"journal\": \"Molecular biomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct 3'-UTR targeting confirmed, glycosylation-localization link established experimentally; single lab, multiple methods\",\n      \"pmids\": [\"39009906\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LYPD3/C4.4A is a GPI-anchored, two-LU-domain cell surface protein (crystal structure resolved) that adopts a compact face-to-face LU-domain arrangement, is heavily N- and O-glycosylated (glycosylation regulates its subcellular localization), binds laminin-1 and laminin-5 as extracellular ligands, and directly interacts with AGR2 (at a site spanning both LU domains) to signal in an autocrine loop requiring laminins and integrin β1; it is cleaved by ADAM10 and ADAM17, recruits activated α6β4 integrin and MT1-MMP into lipid rafts to drive focalized matrix degradation and drug resistance, promotes EMT and cell migration downstream of β2-adrenoceptor signalling, and its transcription requires C/EBPbeta binding to a TRE/CCAAT element cooperating with JunD/c-Jun—collectively establishing LYPD3 as a pro-invasive, pro-metastatic scaffold/adaptor whose loss in knockout mice selectively impairs keratinocyte wound-healing migration and reduces invasive tumor formation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LYPD3 (C4.4A) is a GPI-anchored, two-domain Ly6/uPAR (LU)-module cell surface protein that functions as a pro-invasive, pro-metastatic scaffold coordinating laminin engagement, integrin signalling, and focalized matrix degradation [#3, #8]. Crystallography shows its two LU domains pack face-to-face into a compact globule, atypical for the flexible LU-domain family, with the AGR2-binding site spanning both domains [#13]. It is heavily N- and O-glycosylated, and glycosylation governs its subcellular localization [#1, #15]. Functionally, C4.4A binds the extracellular ligands laminin-1 and laminin-5 and drives spreading, lamellipodia formation, and migration on these substrates [#3]; despite sharing domain architecture with uPAR, it does not bind uPA, marking a distinct functional identity [#2]. At the cell surface it recruits activated alpha6beta4 integrin together with MT1-MMP (MMP14) into lipid rafts to enable localized laminin/matrix degradation and, via alpha6beta4-dependent BAD phosphorylation and Bcl2/BclXl upregulation, drug resistance; this complex persists in exosomes and shed C4.4A retains laminin-degrading activity [#6, #8]. Extracellular AGR2 binds C4.4A directly to drive an autocrine signalling loop requiring laminin-1/5 and integrin beta1 that promotes proliferation, migration, invasion, and chemoresistance [#9]. C4.4A acts upstream of EMT, with knockdown reversing mesenchymal marker expression and reducing invasion across multiple carcinoma types [#7, #11]. Genetic ablation in mice confirms a physiological role: knockout animals develop normally but show delayed keratinocyte wound-closure migration and reduced invasive bladder carcinoma [#10]. Its expression is controlled transcriptionally by C/EBPbeta cooperating with JunD/c-Jun [#4] and post-transcriptionally by miR-151-5p targeting [#15], and it is cleaved by ADAM10 and ADAM17 [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the first candidate binding partners for C4.4A, nominating AGR2/AG-3 and alpha-dystroglycan as interactors and framing it as a partner-engaging surface protein.\",\n      \"evidence\": \"Yeast two-hybrid cloning\",\n      \"pmids\": [\"12592373\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single yeast two-hybrid screen without reciprocal Co-IP validation\", \"Functional consequence of binding not addressed\", \"alpha-dystroglycan interaction not pursued in later timeline entries\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined C4.4A as a GPI-anchored, heavily glycosylated two-LU-module protein and showed it does NOT bind uPA, distinguishing it functionally from its structural homologue uPAR.\",\n      \"evidence\": \"Recombinant protein purification, glycosylation mass spectrometry, and ELISA binding with soluble and GPI-anchored C4.4A\",\n      \"pmids\": [\"15012588\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the functional ligand of C4.4A\", \"Role of the protease-sensitive Tyr200-Arg204 region not tested functionally\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified laminin-1 and laminin-5 as bona fide extracellular ligands and linked C4.4A to cell spreading and migration on laminin, giving the protein a concrete adhesion/motility function.\",\n      \"evidence\": \"ELISA ECM screening, cDNA transfection, migration/spreading assays, and galectin-3 Co-IP\",\n      \"pmids\": [\"15729693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which laminin binding drives migration not resolved\", \"Role of galectin-3 association not mechanistically detailed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved how C4.4A transcription is controlled, showing C/EBPbeta binding to a TRE/CCAAT element cooperating with JunD/c-Jun drives expression.\",\n      \"evidence\": \"Reporter deletion/mutation constructs, EMSA, co-transfection, and ChIP\",\n      \"pmids\": [\"17278103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream signals controlling C/EBPbeta engagement not defined\", \"Single-lab promoter analysis\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified ADAM10 and ADAM17 as the proteases that cleave C4.4A, providing a mechanism for its shedding during invasion.\",\n      \"evidence\": \"SILAC proteomics with shRNA knockdown of ADAM10/ADAM17 in MCF7 cells\",\n      \"pmids\": [\"18979631\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cleavage site and fate of shed fragment not mapped\", \"Functional consequence of shedding inferred rather than directly tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed C4.4A in a lipid-raft complex with alpha6beta4 integrin, MT1-MMP, and TACE that switches cells from laminin adhesion to motility under hypoxia and degrades laminin, including from exosomes.\",\n      \"evidence\": \"Reciprocal Co-IP, wound-healing assays, exosome isolation, and CoCl2 hypoxia model with knockdown\",\n      \"pmids\": [\"22431918\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and assembly order of the raft complex unknown\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Positioned C4.4A upstream of EMT, showing knockdown reverses mesenchymal marker expression and reduces invasion without affecting proliferation.\",\n      \"evidence\": \"siRNA knockdown with EMT marker Western blots and invasion assays in HCT116 cells\",\n      \"pmids\": [\"22404718\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signalling link between C4.4A and EMT transcriptional program not defined\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated in vivo that C4.4A recruits activated alpha6beta4 integrin into rafts to drive focalized matrix degradation and metastasis, while alpha6beta4-BAD-Bcl2 signalling confers drug resistance.\",\n      \"evidence\": \"Stable shRNA knockdown in ASML rat carcinoma cells, intrafootpad metastasis model, raft fractionation, and PI3K/Akt pathway analysis\",\n      \"pmids\": [\"23727360\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of C4.4A-integrin coupling not fully resolved\", \"Single in vivo model system\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Confirmed a direct AGR2-C4.4A interaction and defined an autocrine signalling loop requiring laminins and integrin beta1 that drives PDAC proliferation, invasion, and chemoresistance.\",\n      \"evidence\": \"Co-IP from lysates and recombinant protein mixing, siRNA knockdown, Boyden chamber and apoptosis assays, and orthotopic xenografts with blocking antibodies\",\n      \"pmids\": [\"25646014\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors of AGR2-C4.4A signalling not fully mapped\", \"How the loop integrates with the alpha6beta4/MMP14 raft complex unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided definitive genetic loss-of-function evidence that C4.4A promotes keratinocyte migration and tumor invasion in vivo.\",\n      \"evidence\": \"Constitutive C4.4A knockout mice in incisional wound-healing and chemical bladder carcinogenesis models\",\n      \"pmids\": [\"27169360\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the wound-healing migration defect not dissected in vivo\", \"Normal development indicates functional redundancy not explored\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Generalized the pro-migratory/invasive function to hepatocellular carcinoma and validated C4.4A as an internalizing target amenable to antibody-drug conjugate therapy.\",\n      \"evidence\": \"siRNA knockdown with migration/invasion/proliferation assays in Huh7/HepG2, and ADC (BAY 1129980) efficacy in transfected cells and xenograft/PDX models\",\n      \"pmids\": [\"29048672\", \"28292941\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Internalization route and recycling not characterized\", \"Single-lab functional studies\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Solved the structure of the two LU domains, revealing an atypical compact face-to-face arrangement and mapping the AGR2-binding/antibody epitope spanning both domains.\",\n      \"evidence\": \"X-ray crystallography of C4.4A D1D2 alone and in complex with an anti-C4.4A Fab\",\n      \"pmids\": [\"32140067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length glycosylated C4.4A and its complexes with laminin/integrin not solved\", \"Conformational dynamics in membrane context unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed LYPD3 downstream of beta2-adrenoceptor signalling as a pro-migratory effector in breast cancer.\",\n      \"evidence\": \"Mass spectrometry protein profiling, siRNA knockdown, and migration assays with adrenoceptor agonist/antagonist treatment in MCF-7 cells\",\n      \"pmids\": [\"32098331\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Signalling intermediates linking ADRbeta2 to LYPD3 induction not defined\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established post-transcriptional control of LYPD3 by miR-151-5p (facilitated by METTL3 m6A and hnRNP U) and linked glycosylation to its subcellular localization and a metastasis-suppressive role in HNSCC.\",\n      \"evidence\": \"3'-UTR luciferase reporters, m6A analysis, glycosylation subcellular fractionation, and miR/knockdown functional assays\",\n      \"pmids\": [\"39009906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apparent context-dependent metastasis suppression contrasts with pro-invasive role in other tissues and is unresolved\", \"Glycoform-specific localization mechanism not detailed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the AGR2 autocrine loop, the alpha6beta4/MMP14 raft degradation complex, and the context-dependent pro- versus anti-metastatic roles of LYPD3 are mechanistically integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking signalling input, complex assembly, and tissue-specific outcome\", \"Structure of full-length protein with physiological ligands absent\", \"Direct catalytic activity of LYPD3 itself not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6, 8, 9]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 6, 12]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"complexes\": [\n      \"C4.4A-alpha6beta4 integrin-MT1-MMP/TACE lipid raft complex\"\n    ],\n    \"partners\": [\n      \"AGR2\",\n      \"ITGB4\",\n      \"ITGA6\",\n      \"MMP14\",\n      \"ADAM10\",\n      \"ADAM17\",\n      \"LGALS3\",\n      \"ITGB1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}